The present invention relates to improvements to the enzyme carboxypeptidase G2, and the use of this enzyme in therapy, particularly antibody-directed enzyme prodrug therapy (ADEPT) and gene-directed enzyme prodrug therapy (GDEPT).
Over the years, many cytotoxic compounds have been discovered which are of potential use in cancer chemotherapy. For example, nitrogen mustards form one important family of such cytotoxic compounds. A problem with the clinical use of cytotoxic compounds is in achieving sufficient selectivity in the cytotoxic effect between tumour cells and normal cells. One approach to address this problem has involved the development of so-called prodrugs which are derivatives of the cytotoxic drug, often relatively simple derivatives, whose cytotoxic properties are considerably reduced compared to those of the parent drugs. Numerous proposals have been made for the administration of such prodrugs to patients under regimes whereby the prodrug is only converted by the action of an enzyme to the cytotoxic drug in the region of the intended site of action.
A variety of systems exist for delivery of the enzyme. One such system is described in WO88/07378, and involves conjugating the enzyme to an antibody specific for a tumour marker, delivering the antibody enzyme conjugate to a patient, allowing the conjugate to localise, and then delivering the prodrug to the patient. This system is referred to as antibody-directed enzyme prodrug therapyxe2x80x9d (ADEPT).
Another approach for delivery of the enzyme to the desired site of action is by the use of a genetic construct, such as a viral or non-viral vector carrying a gene encoding the prodrug-converting enzyme, which is delivered to cells at the desired site of action (Huber et al, Proc. Natl. Acad. Sci. USA (1991) 88, 8039). A further alternative system is to provide a ligand, generally a naturally occurring polypeptide whose biological role involves its binding to a cognate receptor on the surface of the cell, conjugated to the prodrug-activating enzyme. This system, LIDEPT, is described in WO/97/26918, where VEGF is particularly exemplified as an example of a ligand. A further alternative system is to use bacterial delivery systems, for example, Clostridium or Salmonella based systems, in which bacteria selectively colonise tumours. A Clostridium based system is described in, for example, Fox et al, 1996 Gene Therapy 3 173-178.
One class of prodrugs suggested for use in the above systems is that of prodrugs of nitrogen mustard compounds. Benzoic acid nitrogen mustards are bifunctional alkylating agents, and a variety of prodrugs of such compounds are described in the art. One class of such prodrugs comprise a protecting group which may be removed by the action of a carboxypeptidase enzyme, such as bacterial carboxypeptidase G, particularly the Pseudomonas-derived enzyme carboxypeptidase G2 (CPG2). CPG2 is a well characterised enzyme with no mammalian equivalent. It is a non-covalently associated, homo-dimeric, metalloenzyme which cleave the C-terminal glutamic acid of folate to yield a pteroate derivative. This has been exploited to cleave glutamic acid from a variety of prodrugs to release potent nitrogen mustard compounds.
Examples of prodrugs which may be activated by CPG2 are described in, for example, Springer et al., Anti-Cancer Drug Design (1991) 6; 467-479, WO88/07378, WO94/25429 and WO96/22277. Fusions of an antibody fragment directed against carcinoembryonic antigen (CEA) with CPG2 have been described in Michael et al, Immunotechnology 2 47-57 (1996) and the use of this fusion in in vivo model systems has been described in Bhatia et al, Int. J. Cancer, 85; 571-577 (2000).
A feature of CPG2 is that being a bacterial enzyme, it does not occur naturally in the body of a human patient, and thus prodrugs designed to be activated by this enzyme will not be activated elsewhere in the patient. However, the drawback to this feature is that the enzyme provokes an immune response in a patient, and indeed such responses have been observed in clinical trials of ADEPT using CPG2 (Sharma et al, 1992, Cell Biophys., 21;109-120; Bagshawe et al, 1995, Tumour Targeting, 1; 17-30).
We have investigated the immunogenicity of CPG2 and identified a number of regions of this enzyme which contain epitopes which appear to be involved in the production of a host immune response. We have found that where a host immune response is caused by the presence of such epitopes, these epitopes may be modified to alter the immunogenicity in patients. However the invention is not limited to this aspect alone, since modifications to these epitopes may be provided which render the CPG2 less reactive with sera from CPG2 immunised patients. The latter aspect of the invention is also advantageous, to allow the development of CPG2 fusions which xe2x80x9cescapexe2x80x9d or xe2x80x9cevadexe2x80x9d, to a greater or lesser degree, an immune response of a host which has been provoked by a wild-type CPG2 or another altered form of CGP2 which has a set of one or more epitope modifications which cause an established host response to a previously administered form of CGP2 to be less effective against the newly altered form.
Throughout this text where specific amino acids or amino acid sequences of the Pseudomonas CPG2 used in the examples below are referred to we have used the numbering used in the Swiss Prot database entry for CPG2 (accession number p06621). The unprocessed form of CPG2 has a sequence 415 amino acids long. The first 22 residues of this sequence are removed in the processed form of CPG2. In the MFE-23::CPG2 fusion protein described here, the first amino acid of the CPG2 domain is amino acid 25 according to the Swiss Prot CPG2 entry.
In a first aspect, the invention provides a CPG2 enzyme in which an immunogenic region selected from:
KIKGRGGK (amino acids 98-105, SEQ ID NO:1)
KEYGVRD (157-163, SEQ ID NO:2), preferably YGVRD (159-163 (SEQ ID NO:6))
KLADY (191-195, SEQ ID NO:3)
GAGK (412-C-terminal(415), SEQ ID NO:4), preferably AG (413-414),
EGGKKLVDK (331-338, SEQ ID NO:5)
is modified to reduce or alter immunogenicity to a mammalian immune system whilst retaining CPG2 activity.
In another aspect, we have also found that production of fusions of CPG2 with an antibody, where the CPG2 protein has been tagged provides a CPG2 protein which has reduced immunogenicity. Thus in a further aspect of the invention, there is provided a CPG2 enzyme, including any of those of the first aspect, which is tagged with a his or myc-his tag.
There is also provided by the invention methods of treatment or diagnosis by methods such as ADEPT, GDEPT or LIDEPT which utilise the CPG2 of the present invention. These and other aspects of the invention are described herein in more detail below.